Dawen Liu

Electroporation optimization to deliver plasmid DNA into dental follicle cells

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 microg/microL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45-120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.

The role of dentin matrix protein 1 (DMP1) in regulation of osteogenic differentiation of rat dental follicle stem cells (DFSCs)

OBJECTIVES Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs. METHODS DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs. RESULTS DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis. CONCLUSIONS DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs.

Proliferation of Dental Follicle Derived Cell Populations in Heat-stress conditions

Objectives:  Isolation and purification of adult stem cells (ASC) are a great challenge. Our objectives were to determine whether ASC are more heat‐tolerant than non‐stem cells, and to explore if ASC could be enriched by heat‐stress treatments.

Expression of endothelial monocyte-activating polypeptide II in the rat dental follicle and its potential role in tooth eruption.

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II contributes to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated on days 1 and 3. Use of a short interfering RNA (siRNA) to knock down EMAP-II expression resulted in a reduction in the expression of colony-stimulating factor-1 (CSF-1) and monocyte chemoattractant protein-1 (MCP-1) in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-(EMAP-II) immunoglobulin G added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in the recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1).

Expression of Bone Morphogenetic Protein-6 in Dental Follicle Stem Cells and Its Effect on Osteogenic Differentiation

The dental follicle (DF) plays an essential role in tooth eruption via regulation of bone resorption and bone formation. Bone morphogenetic protein-6 (BMP6) expression in the DF is coincident with bone growth in the tooth crypt. DF stem cells (DFSCs) have been shown to possess strong osteogenic capability. This study aims to determine the expression of BMP6 in DFSCs and to elucidate the role of BMP6 in the osteogenesis of DFSCs. DFSCs and their non-stem cell counterpart, DF cells (DFCs), were obtained from the DFs of rat pups. We showed that expression of BMP6 was significantly higher in the DFSCs than in the DFCs. DFSCs lost osteogenic capability during in vitro expansion, and DFSCs in late passages had reduced BMP6 expression as compared to early passages of DFSCs when they were subjected to osteogenic induction. Addition of exogenous human recombinant BMP6 (hrBMP6) to the osteogenic medium dramatically enhanced the osteogenesis of the late-passage DFSCs. Knockdown of BMP6 by short interfering RNA in the DFSCs in early passages resulted in a decrease in osteogenesis, which could be restored by addition of hrBMP6. We concluded that DFSCs need to express high levels of BMP6 to maintain their osteogenesis capability. Increased BMP6 expression seen in vivo in the DF may reflect the activation of DFSCs for osteogenic differentiation for bone growth during tooth eruption.

Electroporation to deliver plasmid DNA into rat dental tissues.

Delivery of DNA into the target tissues is an important technique in gene function studies and gene therapy. Surgical treatment of tooth eruption disorders, such as impacted third molars, is a major healthcare cost. Because the dental follicle (DF) is essential for regulating tooth eruption, establishment of local gene transfer protocols is needed to determine the effect of various genes on eruption and to develop gene therapy approaches for inducing the eruption of impacted molars.

Regulation of SFRP-1 expression in the rat dental follicle.

Tooth eruption requires osteoclastogenesis and subsequent bone resorption. Secreted frizzled-related protein-1 (SFRP-1) negatively regulates osteoclastogenesis. Our previous studies indicated that SFRP-1 is expressed in the rat dental follicle (DF), with reduced expression at days 3 and 9 close to the times for the major and minor bursts of osteoclastogenesis, respectively; but it remains unclear as to what molecules contribute to its reduced expression at these critical times. Thus, it was the aim of this study to determine which molecules regulate the expression of SFRP-1 in the DF. To that end, the DF cells were treated with cytokines that are maximally expressed at days 3 or 9, and SFRP-1 expression was determined. Our study indicated that colony-stimulating factor-1 (CSF-1), a molecule maximally expressed in the DF at day 3, down-regulated SFRP-1 expression. As to endothelial monocyte-activating polypeptide II (EMAP-II), a highly expressed molecule in the DF at day 3, it had no effect on the expression of SFRP-1. However, when EMAP-II was knocked down by siRNA, the expression of SFRP-1 was elevated, and this elevated SFRP-1 expression could be reduced by adding recombinant EMAP-II protein. This suggests that EMAP-II maintained a lower level of SFRP-1 in the DF. TNF-α is a molecule maximally expressed at day 9, and this study indicated that it also down-regulated the expression of SFRP-1 in the DF cells. In conclusion, CSF-1 and EMAP-II may contribute to the reduced SFRP-1 expression seen on day 3, while TNF-α may contribute to the reduced SFRP-1 expression at day 9.

MyD88 expression in the rat dental follicle: Implications for osteoclastogenesis and tooth eruption

Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin (IL)-1 and IL-18 toll-like receptor signaling pathways. Because MyD88 is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine expression of the Myd88 gene in vivo in the DFs from the first mandibular molars of postnatal rats from days 1 to 11. The results showed that MyD88 was expressed maximally on day 3. Using small interfering RNA (siRNA) to knock down MyD88 expression in the DF cells also reduced the expression of the nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1) genes. Interleukin-1alpha up-regulated the expression of NFKB1, MCP-1, and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1alpha effect. Conditioned medium from DF cells with MyD88 knocked down had reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis, as opposed to controls. In conclusion, the maximal expression of MyD88 in the DF of postnatal day 3 rats may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression.

Expression of odontogenic ameloblast-associated protein in the dental follicle and its role in osteogenic differentiation of dental follicle stem cells

OBJECTIVE Odontogenic Ameloblast-Associated Protein (ODAM) is encoded by a secretory calcium-binding phosphoprotein cluster gene, which generally plays an important role for mineralization. Dental follicle (DF) is essential in regulating bone formation for tooth eruption. This study aims to reveal ODAM expression in the DFs of developing and erupting molars, and to determine the possible role of ODAM. DESIGN DFs were collected from human third molars and rat mandibular molars for gene expression assessment and for establishment of cell cultures. RT-PCR and western blot were conducted to determine ODAM expression. Over- or silencing expression of ODAM in the dental follicle stem cells (DFSCs) was done by transfecting the cells with ODAM plasmid or siRNA to evaluate ODAM effects on osteogenesis. RESULTS Rat DFs weakly expressed ODAM at early-postnatal days, but a chronological increment of ODAM expression from days 1 to 11 was observed. Differences in expression of ODAM were seen in the human DFs of different individuals. In vitro, ODAM was expressed in DFSCs, but almost no expression in DF-derived fibroblast-like cells. Forcing the DFSCs to overexpress ODAM accelerated osteogenesis, whereas continuously silencing the ODAM in the DFSCs reduced osteogenesis only at 2 weeks of osteogenic induction. CONCLUSIONS ODAM is differentially expressed in the DFs of different age molars. Its expression is coincident with the increased bone formation of tooth crypt during tooth eruption in rat DFs. Increase of ODAM expression may accelerate osteogenic differentiation of DFSCs. Thus, ODAM expression in the DF may regulate bone formation for timely tooth eruption.

Contents Vol. 198, 2013

Founded 1945 as ‘Acta Anatomica’ by R. Chambers, G. Glimstedt, T. Peterfi and G. Wolf-Heidegger Continued 1962–1974 by E.A. Boyden, 1955–1980 by A. Delmas, 1972–1980 by F. Walberg, 1945–1980 by G. Wolf-Heidegger, 1981–1988 by R. O’Rahilly, Davis, Calif., 1989–1990 by G.E. Goslow, Jr., Providence, R.I., 1981–1992 by W. Lierse, Hamburg, since 1992 by H.-W. Denker, Essen, and A.W. English, Atlanta, Ga., continued 1999 as ‘Cells Tissues Organs’ by H.-W. Denker, Essen, and A.W. English, Atlanta, Ga.